Screening for efficient sgRNAs in HEK293 cells
The fourth generation of cytosine base editor (BE4) expresses a Cas9n (D10A) fused to cytidine deaminase (rAPOBEC1) and two copies of uracil glycosylase inhibitor (UGI). To test whether it works in our hand, we first transfected BE4 and pEGFP-sgRNAs in cells that stably express EGFP (Fig. 1A) 9 (Komor et al., 2017). HEK293 cells stably expressing EGFP were generated by transfection of plasmid pEGFP-N1. EGFP expression was checked under a fluorescence microscope. Result showed that up to 90% of cells with fluorescence (Fig. 1D). Positive clones (HEK293-EGFP) were selected using G418 (500μg/mL). Two sgRNAs were designed to target EGFP gene (Fig. 1E, Supplementary Table 1). The specificity score of both sgRNA-1 and sgRNA-2 were 75%, and 83% respectively based on software analysis (https://benchling.com/). HEK293-EGFP cells were co-transfected with BE4 and plasmids encoding sgRNAs (pEGFP-sgRNA1 and pEGFP-sgRNA2). Two days after transfection, EGFP fluorescence intensity was analyzed by fluorescence microscopy (Fig. 1D). Majority of cells transfected with pEGFP-sgRNA1 still express relatively high levels of EGFP (64%), while cells transfected with pEGFP-sgRNA2 exhibited weak signal with an intensity of 36%, indicating EGFP was knocked down more efficiently (Fig. 1D, F). Genomic DNA was extracted for PCR and sequencing to confirm successful base editing.